Poster Presentation The 35th Biennial Conference of the Society of Crystallographers in Australia and New Zealand 2024 (Crystal 35)

Detailed architecture of the human TSC:WIPI3 lysosomal recruitment complex (#208)

Chris Lupton 1 , Charles Bayly-Jones 1 , Laura D'Andrea 1 , Tyler Chang 1 , Gareth Jones 1 , Joel Steele 1 , Hari Venugopal 2 , Michelle Halls 3 , Andrew Ellisdon 1
  1. Biomedical Discovery Institute, Monash University, Melbourne, Victoria, Australia
  2. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, Victoria, Australia
  3. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia

Tuberous sclerosis complex (TSC) regulates cell growth in response to energy stress by inhibiting the master kinase mechanistic target of rapamycin complex (mTORC1). TSC hydrolyzes RAS homolog-mTORC1 binding (RHEB) from its GTP-bound to GDP-bound state, thereby preventing the allosteric activation of mTORC1. Loss-of-function mutations in TSC hyperactivate mTORC1, leading to the common genetic disorder TSC, characterized by excess cell growth and tumor formation. Here, we overcome a high degree of continuous conformational heterogeneity to determine the 2.8 Å cryo-electron microscopy (cryo-EM) structure of the complete human TSC in complex with the lysosomal recruitment factor WIPI3. TSC forms an elongated 40 nm wing-like structure with a core HEAT-repeat scaffold formed by a TSC2 dimer, centrally joined by two juxtaposition catalytic domains. The TSC1 coil-coil dimer runs across the TSC2 surface, forming a previously undetected N-terminal TSC1 dimer that clamps onto the core scaffold on a single TSC wing. Structural and biochemical analysis reveal a novel phosphatidylinositol phosphate (PIP)-binding pocket in the TSC1 dimer interface, which specifically binds singularly phosphorylated PIPs. WD repeat domain phosphoinositide-interacting-protein-3 (WIPI3) binds to the extreme tip of the complex through a conserved motif in TSC1, providing a second membrane anchor point for TSC lysosomal recruitment. These structural advances help explain how TSC and WIPI3 coordinate with endolysosomal PIP-signaling networks to regulate mTORC1 activity at the lysosome. Furthermore, the high-resolution structure of the complete human TSC the identification of novel mutational hotspots and uncover new mechanisms of TSC dysregulation in disease.